The recent progress in molecular biology and its application to medicine, agriculture, and industrial processes has been crucially dependent on structure determinations at the atomic level of macromolecules such as nucleic acids, viruses, large macromolecular complexes and in particular proteins. Redundancies of structural elements now emerging from recently determined protein structures suggests that the number of structural motifs and substructures (domains) naturally occurring in many proteins may be finite, and that these molecular motifs may be classified and catalogued according to specific polypeptide folds. With the majority of protein motifs being determined, structural and functional predictions based primarily on the amino acid sequence may be possible for unknown proteins. With the influx of genomic information presently available, there is more interest being placed in determining the three dimensional structure of proteins encoded by newly identified gene sequences.
The three dimensional structures of proteins has had a dramatic impact on biotechnology and in particular the therapeutic treatment of certain diseases. Determining the three dimensional structure of proteins provides the essential information necessary for identifying and developing new drugs and pharmaceutical products which interact with these proteins (McPherson, Proceedings of Frontiers in Bioprocessing II, (American Chemical Society Boulder, Colo., 1992, pp. 10–15; McPherson, Rational Drug Design, CRC Press, 1994, Chptr. 6, p 170). The three dimensional structure of proteins and other macromolecules is the basis for identifying lead compounds to treat a host of human ailments, veterinary problems, crop diseases in agriculture, and other uses. For example, if the three dimensional structure of the active site of a salient enzyme in a metabolic or regulatory pathway is known, then chemical compounds, such as regulatory drugs, can be rationally designed to inhibit or otherwise affect the behavior of that enzyme.
Another area of biotechnology that also requires knowledge of three-dimensional macromolecular structure is the area of protein genetic engineering. Although recombinant DNA techniques provide the essential synthetic role that permits modification of proteins, protein structure determination provides the analytical function. A three-dimensional protein structure serves as a structural guide or template for intelligent and purposeful protein modifications rather than random and chance amino acid substitutions. Obtaining a three-dimensional structure of the genetically engineered protein is also useful in determining what future chemical and physical enhancement would be profitable endeavors.
Presently, and in the foreseeable future, the only technique that can yield atomic level structural images of biological macromolecules is X-ray diffraction analysis as applied to single crystals. While other methods may produce important structural and dynamic data, for the purposes described above, only X-ray crystallography is adequate. X-ray crystallography is absolutely dependent on crystals of the macromolecule of interest, and not simply crystals, but crystals of sufficient size and quality to permit accurate data collection. The quality of the final structural image is directly determined by the perfection, size and physical properties of the crystalline specimen, hence the crystal becomes the keystone element of the entire process, and the ultimate determinant of its success (McPherson et al., Scientific American, 260 (3) 62–69 (1989).
A considerable degree of success has been achieved recently in the area of macromolecular crystallization. Many new protein structures are rapidly being completed. Unfortunately, some major problems still remain, and in particular, the crystallization of a protein is still the primary obstacle in structure determination. This is particularly true of some major classes of proteins, such as the immunoglobulins, highly dynamic and mobile multidomain molecules, lipophilic molecules, membrane proteins, multimeric protein complexes as well as many small proteins and polypeptides, including hormones.
In the crystal growth of small, non-polymeric molecules, the problem of nucleation is frequently obviated by using seed crystals of the material under study, and in those cases the problems of crystallization arise principally arise from control of growth mechanisms and kinetics. Unfortunately, with macromolecules and proteins whose structures remain undetermined, simple seeding is not possible due to the lack of the availability of preexisting crystals. When a protein is being crystallized for the first time, nucleation is of particular significance.
One aspect of crystal growth, which has been useful in the nucleation of macromolecules, is the recognition that surfaces that contact the solution (mother liquor) from which crystals are formed, are of substantial importance in accelerating the nucleation process. Molecules, and especially macromolecules having extensive surfaces with diverse charge distributions, adsorb to, and are concentrated at surfaces in contact with their solutions. In addition, many surfaces, because of their own charge properties and microstructure, help to organize the macromolecules into more ordered assemblies and unit structures. Such surfaces thus serve to promote the formation of critical nuclei. Thes surfaces tend to act as catalysts to lower the activation energy for macromolecular nucleation and increase the probability that nuclei will form to initiate the crystallization process.
One method of crystal growth has been developed where the electrical state of the nucleant has been shown to encourage macromolecular nucleation (see, e.g., U.S. Pat. No. 6,110,273 and U.S. Pat. No. 6,258,331). In this method, a substrate with a layer of n-type silicon on p-type silicon provides adjacent layers of contrasting electrical properties. The substrate is patterned lithographically using a photoresist to defme grooves or channels of varying depths and with different cross-sectional shapes or patterns. Valence electrons are controlled in the substrate such that the concentration of holes or electrons in the substrate surface can be controlled in response to the environment of the solution (containing the macromolecule) that is exposed to the substrate. The valence electron control facilitates crystallization of the macromolecule inside, rather than outside, of the grooves in the substrate.
There has also been an effort to develop a crystal growth apparatus that facilitates the crystallization of biological macromolecules. One such apparatus involves a liquid reservoir for crystal growth, with passages for transferring the liquid from the first reservoir to a second liquid reservoir. The first reservoir holding the liquid for growing crystals is made of general-purpose material such as glass while the second liquid reservoir is formed on a doped silicon substrate. The material of the second reservoir has valence electrons that are controllable so that the concentration of holes or electrons can be controlled in response to the environment of the solution containing the macromolecule. This allows for the crystals to be grown on a silicon substrate that has been placed in a predetermined electric state.
One of the most serious problems in protein crystallization is the high supersaturation concentrations of the protein which are required for spontaneous nucleation. These high supersaturations are often above the solution stability of the protein, resulting in the formation of numerous small, disordered, unusable crystals instead of a few large, well diffracting, x-ray quality crystals. One crystallization technique that avoids the need for high supersaturation concentrations involves the use of heterogeneous nucleants for protein crystal growth. Shlicta et al., J. Crystal Growth 174 (1997) 480–486, reported the effect of heterogeneous substrates to promote protein crystallization at lower protein supersaturation concentrations. In this study, inorganic substrates were utilized to grow protein crystals. These inorganic substrates had a far smaller unit cell than the proteins examined but gave a precise lattice match. Shlicta et al. have prepared a database for lattice-matches for the inorganic substrates tested which can help expidite the search for candidate exptiaxial substrates. Unfortunately, for this database to be useful, some structural information about the protein to be crystallized must still be known, (e.g. unit cell dimensions).
“Heterogeneous nucleants” such as are provided by some mineral surfaces, have served as efficient nucleants for a number of different proteins (McPherson et al., Crystal Growth 85 (1987) 206–214). McPherson et al. studied such minerals as apophyllite and magnesium oxide, which were capable of inducing nucleation of lysozyme, concavalin and bovine catalase. McPherson et al. (Science (1988) 238, pp. 385–387) have also investigated the heterogeneous and epitaxial nucleation of protein crystals using a combinatorial library of solid state inorganic materials having a variety of compositions of varying crystallinity and structure types. McPherson et al. demonstrat the use of heterogeneous nucleants to promote epitaxial growth of proteins. They demonstrated the heterogeneous nucleation of proteins on mineral surfaces can occur at lower levels of critical supersaturation, but that true epitaxial growth of protein on mineral crystals can also occur. They rationalized that other ordered two-dimensional arrays, as obtained from synthetic materials, might serve as nucleants as well.
McPherson et al., J. Crystal growth, 85 (1987) 206–2140, also investigated the facilitation of the growth of protein crystals by heterogeneous/epitaxial nucleation by using inorganic crystals for the nucleation of protein crystals. The four proteins crystallized were examined by vapor diffusion techniques in the presence of 50 species of inorganic crystals employed as a “combinatorial library” of different surfaces. This study demonstrated that a substantial decrease in critical supersaturation was caused by certain nucleants for some of the proteins tested which facilitated nucleation of protein crystal growth.
U.S. Pat. No. 5,869,604 discloses the use of an exogenous nucleating agent having a close lattice match to a polypeptide of interest, for nucleation and growth of high purity polypeptide crystals. In order for effective nucleation, the lattice dimensions of the crystal face of the nucleating agent must substantially match an integral multiple of a unit cell component of the polypeptide of interest. This sort of lattice matching is difficult or impossible without some structural knowledge of the polypeptide. Thus, there has been far more interest in use of non-epitaxial heterogeneous nucleants, mainly because these do not require any prior knowledge of a protein's unit cell constants.
Determining the correct conditions for crystallization of biological molecules can be labor intensive, and large quantities of biological material may be needed which may not be readily available. There have been recent efforts to develop methods for screening crystallization conditions which minimize the overall amount of biological material utilized as well as lend themselves to high throughput crystallization using picogram to microgram quantities of proteins (see, e.g., WO 00/60345). One such method allows for the screening of multiple samples simultaneously, each screening sample having picogram to microgram amounts of protein in a picoliter to nanoliter volume. These micro-chambers containing the protein may be passive, or a combination of passive micro-chambers that are connected with miniaturized active control elements such as valves, pumps and electrodes. This type of protein crystallization apparatus allows for a protein solution to be automatically dispensed into the micro-chambers, where each micro-chamber contains a slightly different crystal growth condition. Protein crystal growth in the micro-chambers is then analyzed based on both the qualitative amount of crystallization and the quality of the crystals formed. Unfortunately, use of this type of apparatus and method is labor intensive, since a different solution must be added into each micro-chamber.
The development of suitable heterogeneous substrates effective in promoting crystallization of a wide variety of proteins would provide substantial advantages in the field of protein X-ray crystallography. Any ffort at carrying out high throughput crystallization of macromolecular structures will unquestionably encounter the basic and crucial obstacle of crystallization. The other elements of the high throughput efforts, such as target selection and expression, protein purification, data collection and structure solution, have all received far more attention than the actual critical and essential crystallization event. Effective heterogeneous substrates for protein and macromolecule crystallization, however, have heretofore been unavailable.
Thus there is a need for apparatus and methods for rapid and assured crystallization of target macromolecules as well as apparatus and methods which can be automated to allow for high throughput screening of crystallization conditions for one or more molecules in parallel. There is also a need for a crystallization apparatus and methods which require minute or minimal amounts of protein for inducing nucleation. The present invention satisfies these needs, as well as others, and overcomes the deficiencies found in the background art.